The aberrant over expression and concomitant activation of members of the ErbB family of growth factor receptor tyrosine kinases plays key roles in promoting the growth and progression of a variety of solid tumor types. The long term objective of the proposed studies is to understand the role of a novel protein degradation pathway in regulating ErbB3-induced mammary tumor initiation and progression. The central component of the pathway is a RING finger E3 ubiquitin ligase called Nrdp1 that mediates the ubiquitination of the ErbB3 receptor, thereby promoting its trafficking to degradative cellular compartments. Nrdp1 protein is invariably post-transcription ally suppressed in ErbB2-induced mammary tumors from transgenic mice that over express endogenous ErbB3, and Nrdp1 protein is lost in tumors from almost 60% of breast cancer patients, consistent with a model whereby Nrdp1 loss facilitates ErbB3 protein over expression in tumors. However, recent evidence points to a broader role for Nrdp1 and its associated pathway components in cellular regulation. The hypothesis guiding the proposed studies is that Nrdp1 acts through multiple pathways to maintain mammary epithelial cell integrity, and that the post-transcriptional loss of Nrdp1 protein removes a barrier to mammary tumor initiation. Three critical issues concerning Nrdp1 involvement in mammary epithelial integrity will be addressed. 1) The role of the Nrdp1 pathway-associated deubiquitinating enzyme Otub1 in mediating Nrdp1 post-transcriptional loss in tumor cells will be assessed by viral-mediated over expression and knockdown in cultured cells. In addition, inducible transgenic over expression of Otub1 in the mammary gland will be used to assess its involvement in mammary gland development and ErbB2-induced tumorigenesis. 2) The role of Nrdp1 pathway components in establishing and maintaining the polarity and architecture of cultured epithelial cells will be explored using viral-mediated over expression and knockdown approaches coupled with biochemical assays and immunofluoresecence-based cell structure assays. 3) The role of the Nrdp1 gene in suppressing the onset of ErbB2-induced tumors will be examined using conditional knockout and transplantation approaches. Together, the proposed studies could uncover novel activities for this pathway in mammary tumor development, and underscore its role in mammary tumor suppression. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding molecular and cellular mechanisms underlying breast cancer initiation and progression. The successful completion of the work could shed light on the biochemical pathways engaged by normal or pre-malignant breast epithelial cells during their transition to a malignant state. Ultimately, the studies could identify novel targets for therapeutic intervention.